Globalization has provided opportunities for parasites/pathogens to cross geographic
boundaries and expand to new hosts. Nosema disease is one of the most
serious adult honey bee diseases and has high prevalence in honey bee colonies. For years,
Nosema apis was thought to be the only microsporidian infecting
domestic bee colonies. However, recently it was discovered that N. ceranae
could cross the species barrier from Asian honey bees (Apis
cerana) to European honey bees (Apis mellifera) that are
widely used for crop pollination and honey production. Over the last few years,
considerable progress has been made in our understanding of Nosema
infections in honey bees. This review summarizes previous findings and recent
progress in the understanding of Nosema infection of A. mellifera
in the USA and Asia, with particular emphasis on the comparative epidemiological,
morphological, pathological, and genomic organization of two Nosema
species. The prospects of future research and remaining unresolved questions
associated with the study of honey bee Nosema diseases are also
discussed.

1. INTRODUCTION

Nosemosis (Nosema disease) is one of the most serious and prevalent adult
honey bee diseases worldwide (Bailey, 1981; Matheson,
1993; Fries, 2010) and is caused by intracellular microsporidian parasites from genus of
Nosema. For decades, Nosema disease was exclusively
attributed to a single species of Nosema, N. apis, which
was first described in European honey bees, Apis mellifera (Zander, 1909). In 1996, a new species of Nosema
was first discovered in the Asian honey bee, Apis cerana, thus
named Nosema ceranae (Fries et al., 1996). In 2005, a natural infection of N. ceranae was reported in
A. mellifera colonies from Taiwan (Huang et al., 2005). Shortly thereafter, the infection of N. ceranae
to A. mellifera was reported in Europe (Higes et al., 2006; Paxton et al., 2007), United States (Chen et al., 2007),
China (Liu et al., 2008), Vietnam and worldwide (Klee
et al., 2007). Since its emergence as a potentially
virulent pathogen of A. mellifera, N. ceranae has been
associated with colony collapse of honey bees (Higes, et al., 2008; Paxton, 2010). A recent
study showed that N. ceranae expanded its host range to South American
native bumblebees (Plischuk et al., 2009) causing a
new epidemiological concern for this pathogen. The present review summarizes recent findings
on Nosema ceranae infection of A. mellifera in the USA and
Asia, with particular emphasis on the comparative epidemiological, morphological,
pathological, and genomic analysis of two Nosema species.

2. THE PREVALENCE OF NOSEMA INFECTION IN THE UNITED STATES

A study for screening the prevalence of Nosema infections in the USA
population of A. mellifera was conducted in 2007 (Chen et al., 2007). Bee samples collected between 1995 and 2007 from
different geographic regions of the USA were examined individually for the presence of both
N. apis and N. ceranae using the PCR method. The results
showed that N. ceranae had a wide-spread infection of A. mellifera
in the USA. N. ceranae infected bees were found in samples
collected from each of 12 states including Oregon, California, Hawaii, Idaho, North Dakota,
Minnesota, Texas, Ohio, Tennessee, Connecticut, Maryland and Florida, representing the
Northeast, Southeast, Midwest, Southwest, and the West regions of the USA Among the 180 bees
examined for Nosema, 16% of the bees were positive for N. ceranae,
while N. apis was not detected. The absence of N. apis
may have been caused by inadequate sampling. The detection of N. ceranae
in honey bees collected in 1995 indicated that N. ceranae is not a
new emerging pathogen for A. mellifera in the USA and, in fact, had
transferred from its presumed original host A. cerana at least a decade
ago. Although the data presented in this study demonstrated that N. ceranae
infection was widespread in the USA, the authors believed that distribution of
N. ceranae infection of A. mellifera could be even more
widespread than had been identified, if a more intensive epidemiological investigation was
conducted. Later work by Williams et al. (2008)
detected infection of N. ceranae in honey bees from the Maritime Provinces
of Canada and Minnesota, USA and expanded the known distribution of this parasite.

While Chen et al. (2007) reported that PCR
amplification using N. apis specific primers did not yield any positive
results for bee samples tested, a study by the consortium scientists using a metagnomic
approach to survey microflora in Colony Collapse Disorder (CCD) affected bee colonies and
healthy colonies showed that co-infections of N. apis and N.
ceranae were found in A. mellifera, and that the infection rate
of N. ceranae was significantly higher than that of N. apis
in bees from both CCD affected colonies and normal healthy colonies (Cox-Foster et
al., 2007). A similar result was obtained from a more
recently conducted CCD descriptive epidemiological study (vanEngelsdorp et al., 2009). The studies showed that the infection rate of
N. ceranae was 55% and 50% in CCD and control colonies, respectively,
while the infection rate of N. apis was 29% and 18% in CCD and control
colonies, respectively. All of these results were in line with a previous report that prior
to 2003 most bee samples had N. apis infection but N. ceranae
became a predominant infection after 2003 (Klee et al., 2007). The studies conducted in the USA confirm and extend early
observations by Fries et al. (2006), Higes et al.
(2006, 2007)
and Huang et al. (2007) that N. ceranae
was not restricted to its original host, but has established an infection in the
European honey bee for some time, and that infection with N. ceranae is now
more common than infection with N. apis in European honey bees.

3. THE PREVALENCE OF NOSEMA INFECTION IN EAST ASIA AND AUSTRALIA

A survey for the infection of A. mellifera with both N. ceranae
and N. apis was performed in China (Liu et al., 2008). The samples of honey bees were collected from 12
different apiaries located in ten provinces and two municipalities in China. Thirty bees
from each apiary were pooled together and examined for the presence of N. ceranae
and N. apis using the PCR assay (Liu et al., 2008). N. ceranae were found to be present in every
apiary examined. Sequence comparison of PCR fragments generated from the study with
published sequences at the GenBank resulted in 99% sequence identity for N. ceranae
and confirmed the specificity of the PCR assay. No N. apis was
detected in any samples examined.

In contrast to the finding in the USA and China that N. ceranae was
identified as the sole or predominant infection in A. mellifera, bee
samples from Australia showed a notably higher rate of N. apis infection
(46.3%) than N. ceranae infection (15.3%) (by calculation from Table 2,
Giersch et al., 2009). While N. ceranae
was detected in samples collected from only four states (Queensland, New South
Wales, Victoria, and South Australia), N. apis was found in samples
collected from every state. Among the 307 bees examined for infection, only two bees had
co-infection of both Nosema species. Further, the prevalence of N.
ceranae infection varied considerably across states. While Western Australia and
Tasmania were found to have no incidence of N. ceranae infection,
N. ceranae was detected in 33.7%, 16%, 15.8%, and 4.5% of bees collected
in Queensland, South Australia, New South Wales, and Victoria, respectively. The honey
samples that originated from beekeepers in Queensland were also PCR positive for N.
ceranae (Giersch et al., 2009). The
infection of N. ceranae and N. apis in Australian
population of A. mellifera obviously constitutes a unique case of
Nosema prevalence compared to other reported cases from other regions of
the world (Chen et al., 2007; Higes et al., 2007; Klee et al., 2007; Liu et al., 2008). One hypothesis is
that N. ceranae in Australia may have a relatively recent introduction
compared to other regions of the world. Queensland had the highest rate of N.
ceranae infection among all the states and, therefore, may represent the region
with the longest history of N. ceranae establishment. Alternatively, the
variation in Nosema prevalence may also be due to different climate
conditions in different geographical regions (Giersch et al., 2009).

Fries and Feng (1995) first reported that N.
apis can infect A. cerana under laboratory conditions. A recent
study conducted by Chen et al. (2009b) confirmed that
this is also true under natural conditions. Samples of A. cerana collected
from China, Japan and Taiwan showed that both N. apis and N.
ceranae were present as single or as co-infections in Asian honey bees. However,
N. ceranae was the significantly more common infection of the two
Nosema species as N. apis was detected in 31% of
examined bees while N. ceranae was detected in 71% of examined bees.
Quantification of Nosema by real time quantitative PCR showed that the copy
number of N. ceranae was 100 times higher than the copy number of
N. apis in coinfected bees (Chen et al., 2009b). The study indicates that host shifting also occurred for N. apis,
in that N. apis not only attacks European honey bees but also
Asian honey bees and that N. ceranae is also the more common and
predominant infection of the two Nosema species in Asian honey bees.

4.1. Morphology

The morphological and developmental features of N. ceranae have been
described by Fries et al. (1996, 2006), Higes et al. (2007) and Chen et al. (2009a) and the
results from different work groups were generally similar. By light microscopy, fresh
N. ceranae spores were oval or rod shaped, measuring 4.4 ±
0.41 μm (mean ± SD) in length and 2.2 ± 0.09 μm (mean ±
SD) in width (Chen et al., 2009a). Compared to the
spores of N. apis with 6.0 μm in length and 3.0 in width
(Fries et al., 1996), the size of N.
ceranae spores is smaller than that of N. apis spores. By
electron microscopy, N. ceranae displayed all of the ultrastructural
features of the genus Nosema including (1) diplokaryotic nuclei present
in all developmental stages, (2) a long flexible polar filament that appears in the mature
spores, (3) meronts, the earliest stages in the life cycle of the parasite, which are in
direct contact with host cell cytoplasm, (4) mature spores that are bounded by a thickened
wall consisting of electron-dense exospore and electron-lucent endospore layers, and (5)
the thickness of exospore that is 48–52 nm, within the range of 40–60 nm in the genus
Nosema (Larsson, 1986). The
longitudinal section of a mature spore demonstrates the similarity of internal
ultrastructures between N. ceranae and N. apis (de Graaf
et al., 1994). The lamellate polaroplasts right
below an anchoring disc and the posterior vacuole are located in the anterior and
posterior ends of the spore, respectively. Each spore contains a coiled polar filament,
surrounding the diplokaryon. The number of coils of polar filament inside N.
ceranae spores was 18 to 21 (Chen et al., 2009a). Compared to N. apis which has more than 30 coils
(Fries, 1989; Liu, 1984), N. ceranae has a smaller number of coils in the polar
filament. The difference in the size of spores and the number of polar filament coils
provides evidence of morphological differences between N. ceranae and
N. apis.

The tissue tropism (affinity to specific tissues) of a parasite is an important
pathogenic factor. Infection of Nosema starts through ingestion of spores
with food or water. Following ingestion, the spores develop in the site of the primary
infection and multiplied parasites can spread to different tissues of the same host. A
study conducted by Chen et al. (2009a) using PCR
method showed that N. ceranae has a broad tissue tropism in the host of
A. mellifera. The infection of N. ceranae was not
restricted to the midgut tissue but spread to other tissues including the malpighian
tubules, hypopharyngeal glands, salivary glands, and fat bodies (Fig. 1). Among bee tissues dissected and examined, N. ceranae
was detected in 100% of alimentary canals, malpighian tubules, and hypopharyngeal
glands, in 87% salivary glands, and in 20% of the fat bodies. No N.
ceranae-specific PCR signal was detected in the muscle tissue. The infection of
Nosema in European honey bees has often been reported to be associated
with effects of reduced bee longevity, decreased population size, higher autumn/winter
colony loss, reduced honey production and decreased brood production (Hassanein, 1953a, b; Rinderer and Sylvester, 1978; Anderson and Giacon, 1992;
Goodwin et al., 1990; Malone et al., 1995). However, none of the disease symptoms such as
dysentery and/or crawling behavior and/or milky white coloration of gut that are usually
related with N. apis infection has been found in N. ceranae
infected bees (Fries et al., 2006). It was
shown recently that N. ceranae exerts a significant energy cost to
infected bees and changes their feeding behavior (Mayack and Naug, 2009; Naug and Gibbs, 2009). An
early study by Bailey and Ball (1991) demonstrated
that infection of hypopharyngeal glands by N. apis could lead to worker
bees losing the ability to produce brood food and digest food The absence of crawling
behavior in N. ceranae infected bees might be the result of absence of
N. ceranae infection in the muscles. Fat body is one of the primary
sites of microsporidian infection in many insects. The infection of adipose tissue causes
formation of whitish cysts and the infected gut becomes swollen and whitish as a result of
impaired fat metabolism (Sokolova et al., 2006).
The absence of milky white coloration of gut may reflect low infection of N.
ceranae in the tissue of the fat body. Because all previous tissue tropism
studies on N. apis were conducted using the presence of spores as a
criterion (Hassanein, 1953a, b; Gilliam and
Shimanuki, 1967; de Graaf and Jacobs, 1991), new efforts are under way as part of a recently
funded USDA-CAP project to determine the tissue tropism of N. apis in the
host of A. mellifera (Lee Solter, unpubl. data). While N. apis
was known to cause earlier foraging in A. mellifera (Hassanein,
1953; Wang and Moeller, 1970), this behavioral change seems to be mediated by higher juvenile
hormone titers in infected bees due to elevated juvenile hormone production (Huang, 2001), comparative data is lacking in N.
ceranae. Further studies on the pathogenesis of both parasites will shed light
on why N. ceranae has different pathological effects on the host of
A. mellifera compared to N. apis.

4.3. Ribosomal RNA secondary structure models

Secondary structure models for the large subunit ribosomal RNA (LSUrRNA) of
N. ceranae and N. apis. The structure models of
LSUrRNA of N. ceranae and N. apis are
identical.

Secondary structure refers to a folded, three-dimensional configuration of RNA based on
the primary sequence of RNA. For RNA molecules, the secondary structure is more important
for their biological functions than their primary sequences. Knowing the secondary
structures can help to gain a deeper insight into the biological activities of the
parasite in the host. A comparative sequence analysis was conducted to predict small
subunit ribosomal RNA (SSUrRNA) and large subunit rRNA (LSUrRNA) secondary structures for
both N. ceranae and N. apis based on complete sequences
of ribosomal genes of both species first deposited in GenBank. The complete DNA sequences
of the ribosmomal RNA gene of N. ceranae contained 4475 bp (GenBank
accession number DQ486027). The DNA sequence of the SSUrRNA cistron was located at the 5′
end between nucleotide 1–1259. The G+C content of the SSUrRNA cistron was 36.46%. The
internal transcribed space (ITS) region consisted of a 39 bp sequence and was located
between nucleotides 1260–1298. The DNA sequence of LSUrRNA contained 2530 bp and was
located at the 3′ end between nucleotide 1299–3828. The GC composition of the N.
ceranae LSUrRNA sequences was 32.86% (Chen et al., 2009a). The complete DNA sequences of the rRNA gene of N. apis
contained 3756 bp (GenBank accession number U97150). The DNA sequence of the
SSUrRNA cistron was located at the 5′ end (1242 bp) while the DNA sequence of the LSUrRNA
was located at the 3′ end (2481 bp). Both SSUrRNA and LSUrRNA were separated by an ITS
(33bp). The DNA sequence is also presented for the regions flanking the 5′ end of the
small subunit gene and the 3′ end of the large subunit gene (Gatehouse et al., 1998). As shown in Figure 2 and 3, comparative structural models of
SSUrRNA and LSUrRNA indicate that ribosomal RNAs of N. ceranae and
N. apis are conserved and contain all of the structural features that
are characteristic of known microsporidian rRNAs (Figs. 3 and 4) (Gutell et al., 1986a, b). While the microsporidian rRNAs contain some
of the characteristic features found in the vast majority of the eukaryotic rRNAs, the
SSUrRNA and LSUrRNA of N. ceranae and N. apis are very
unusual. They lack many of the structural elements present in other nuclear-encoded
eukaryotic rRNAs, and are significantly shorter in length. For example, the SSUrRNA and
LSUrRNA of Saccharomyces cerevisiae, a species of budding yeast, are
approximately 1800 and 3550 nucleotides in length respectively. The SSUrRNA of N.
ceranae and N. apis are 1259 and 1242 bp nucleotide in length,
respectively, while the LSUrRNA of N. ceranae and N. apis
are 2530 and 2481 nucleotides in length, respectively. Further studies are needed
to determine how the reduction in size of rRNA contributes to the life cycle of the
intracellular parasite in the host.

4.4. Phylogenetic analysis

A phylogenetic analysis of 20 species of microsporidia with highest BLAST score to
N. ceranae was conducted with their sequences of SSUrRNA. Although
N. apis and N. ceranae infect the same host and share
similarities in sequences of rRNA gene, phylogenetic analysis showed that N. apis
is not the closest relative of N. ceranae. Within the same
clade, N. ceranae appears to be more closely related to N.
vespula, a parasite infecting wasps. N. apis seems to have
branched off earlier in evolution and is most closely linked to N. bombi,
a parasite infecting bumble bees (Chen et al., 2009a) (Fig. 5). This result is in agreement
with the earlier phylogenetic work by Fries et al. (1996). The result obtained from Nosema phylogenetic analyses
indicates that parasites from the same host species are not necessarily more closely
related to each other and that evolutionary relationship is not always based on the host
specificity of the taxa. The evolutionary distance between N. ceranae and
N. apis may explain their difference in the morphological features and
tissue specificities in the host.

Secondary structure models for the SSUrRNA of N. ceranae and
N. apis. The structure models of SSUrRNA of N. ceranae
and N. apis are identical in general except there are two
extra loops present in the secondary structure of SSUrRNA of N. ceranae
(highlighted by boxes) compared with structure of N.
apis.

4.5. Genome-wide sequencing and analysis

The complete genome of N. ceranae was recently sequenced using 454
sequencing approach (Cornman et al., 2009). The
sequence information and annotations of N. ceranae are posted in GenBank
under Genome Project ID32973. Pyrosequence data of N. ceranae lead to a
draft assembly and annotated genome of 7.86 Mbp. N. ceranae has a
strongly AT-biased genome, with 74% AT content and a diversity of repetitive elements. The
initial sequencing and assembly of N. apis lead to a genome size of 6–9
Mbp with a GC content of less than 20%. Like N. ceranae, N. apis
also has a strongly AT-riched genome (unpublished data). The genome sequence
project of N. apis has just reached the stage of assembly and annotation.

The computational analysis of genomic sequence data of N. ceranae led to
identification of 2641 putative protein-coding genes. A comparative genomics analysis of
2641 N. ceranae genes with those of another fully sequenced
microsporidian, Encephalitozoon cuniculi, and with the yeast, S.
cerevisiae, showed that N. ceranae has 1252 (48%) orthologous
genes in E. cuniculi and 466 (18%) orthologous genes in S.
cerevisiae. Of the 2614 predicted protein-coding sequences, there are only 11
genes that are both well-conserved and found only in microsporidia and lack clear homology
outside this group. Future comparisons of the genes conserved among microsporidia in these
two Nosema species will provide valuable insights and tools for
identifying virulence factors in this group of the parasites.Mapping individual genes to
standard metabolic pathways has provided important insights into the metabolic pathway in
N. ceranae. A unique feature of microsporidia is that they do not have
distinct mitochondria, a cell organell for generating energy, during the evolution and
thus utilize the host ATP for their energy metabolism. The identification of metabolic
’chokepoints’ of N. ceranae would be especially attractive targets for
chemical or genetic control strategies.

Phylogenetic tree of microsporidia infecting insects based on the sequences of the
SSUrRNA gene. Trachipleistophora hominis infecting Homo
sapiens was used as an outgroup. The tree was constructed by Maximum
Parsimony analyses under a heuristic search. The reliability of the tree topology
was determined by the bootstrap analysis (1000 replicates). The bootstrap values are
located on the tree branches.

5. CONCLUSION

The finding about the prevalence of N. ceranae in the USA and Asian bee
populations in conjunction with previous findings in Europe and other parts of the world
raises several questions regarding N. ceranae infection in European honey
bees. First, when was the exact time that N. ceranae expanded its host
range from A. cerana to A. mellifera? Which transmission
pathway(s) provided opportunities for N. ceranae to overcome the species
barriers to expand its host range and establish infection in a new host? What mechanisms
underlying virulence of N. ceranae led to N. ceranae
becoming the more prevalent infection of the two Nosema species in
A. mellifera? What physiologic and genetic characteristics of the host
are favored by N. ceranae and contribute to determining host range
expansion? All of these questions indicate a strong need for further investigation of the
evolutionary history and molecular mechanisms of pathogenesis of N. ceranae
in European honey bees. The availability of genomic information of two
Nosema species will definitely enhance our understanding of the
evolutionary history and disease mechanism of Nosema in the host. The
comparative genomic analysis of N. ceranae and N. apis
will provide valuable insights and tools for identifying genes that are conserved
between two Nosema species and genes that are responsible for the
successful parasitism and major epidemics of N. ceranae in honey bees. The
genomic information will also enable the researchers to develop and use genetic markers to
seek a better understanding of the epidemiology of Nosema infections and
pinpoint the signals that control gene function, which in turn should translate into new
strategies for combating Nosema disease and improving honey bee health.

Acknowledgments

We thank Dr. Robin Gutell from University of Texas for the help of the secondary structure
models of ribosomal RNA. The research was supported in part by a USDA NIFA AFRI grant
“Managed Pollinator Coordinated Agricultural Project” (2009-85118-05718) and a USDA NRI grant
(97-35302-5304).

Secondary structure models for the SSUrRNA of N. ceranae and
N. apis. The structure models of SSUrRNA of N. ceranae
and N. apis are identical in general except there are two
extra loops present in the secondary structure of SSUrRNA of N. ceranae
(highlighted by boxes) compared with structure of N.
apis.

Phylogenetic tree of microsporidia infecting insects based on the sequences of the
SSUrRNA gene. Trachipleistophora hominis infecting Homo
sapiens was used as an outgroup. The tree was constructed by Maximum
Parsimony analyses under a heuristic search. The reliability of the tree topology
was determined by the bootstrap analysis (1000 replicates). The bootstrap values are
located on the tree branches.